The TGF-β superfamily includes five distinct forms of TGF-β (Sporn and Roberts (1990) in Peptide Growth Factors and Their Receptors, Sporn and Roberts, eds., Springer-Verlag: Berlin pp. 419-472), as well as the differentiation factors Vg-1 (Weeks and Melton (1987) Cell 51: 861-867), DPP-C polypeptide (Padgett et al. (1987) Nature 325: 81-84), the hormones activin and inhibin (Mason et al. (1985) Nature 318: 659-663; Mason et al. (1987) Growth Factors 1: 77-88), the Mullerian-inhibiting substance, MIS (Cate et al. (1986) Cell 45:685-698), osteogenic and morphogenic proteins OP-1 (PCT/US90/05903), OP-2 (PCT/US91/07654), OP-3 (PCT/WO94/10202), the BMPs, (see U.S. Pat. Nos. 4,877,864; 5,141,905; 5,013,649; 5,116,738; 5,108,922; 5,106,748; and 5,155,058), the developmentally regulated protein Vgr-1 (Lyons et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4554-4558) and the growth/differentiation factors GDF-1, GDF-3, GDF-9 and dorsalin-1 (McPherron et al. (1993) J. Biol. Chem. 268: 3444-3449; Basler et al. (1993) Cell 73: 687-702), to name but a few.
The proteins of the TGF-β superfamily are disulfide-linked homo- or heterodimers that are expressed as large precursor polypeptide chains containing a hydrophobic signal sequence, a long and relatively poorly conserved N-terminal pro region sequence of several hundred amino acids, a cleavage site, a mature domain comprising an N-terminal region that varies among the family members and a highly conserved C-terminal region. This C-terminal region, present in the processed mature proteins of all known family members, contains approximately 100 amino acids with a characteristic cysteine motif having a conserved six or seven cysteine skeleton. Although the position of the cleavage site between the mature and pro regions varies among the family members, the cysteine pattern of the C-terminus of all of the proteins is in the identical format, ending in the sequence Cys-X-Cys-X (Sporn and Roberts (1990), supra).
A unifying feature of the biology of the proteins of the TGF-β superfamily is their ability to regulate developmental processes, including endochondral bone morphogenesis. These structurally related proteins have been identified as being involved in a variety of developmental events. For example, TGF-β and the polypeptides of the inhibin/activin group appear to play a role in the regulation of cell growth and differentiation. MIS causes regression of the Mullerian duct in development of the mammalian male embryo, and dpp, the gene product of the Drosophila decapentaplegic complex, is required for appropriate dorsal-ventral specification. Similarly, Vg-1 is involved in mesoderm induction in Xenopus, and Vgr-1 has been identified in a variety of developing murine tissues. Regarding bone formation, proteins in the TGF-β superfamily, for example OP-1 and a subset of other proteins identified as BMPs (bone morphogenic proteins) play the major role. OP-1 (BMP-7) and other osteogenic proteins have been produced using recombinant techniques (U.S. Pat. No. 5,011,691 and PCT Application No. US 90/05903) and shown to be able to induce formation of true endochondral bone in vivo. The osteogenic proteins generally are classified in the art as a subgroup of the TGF-β superfamily of growth factors (Hogan (1996), Genes & Development, 10:1580-1594).
Recently certain members of this same family of proteins have been recognized to be morphogenic, i.e., capable of inducing the developmental cascade of tissue morphogenesis in a mature mammal (See PCT Application No. US 92/01968). In particular, these morphogens are capable of inducing the proliferation of uncommitted progenitor cells, and inducing the differentiation of these stimulated progenitor cells in a tissue-specific manner under appropriate environmental conditions. In addition, the morphogens are capable of supporting the growth and maintenance of these differentiated cells. These morphogenic activities allow the proteins to initiate and maintain the developmental cascade of tissue morphogenesis in an appropriate, morphogenically permissive environment, stimulating stem cells to proliferate and differentiate in a tissue-specific manner, and inducing the progression of events that culminate in new tissue formation. These morphogenic activities also allow the proteins to induce the “redifferentiation” of cells previously stimulated to stray from their differentiation path. Under appropriate environmental conditions it is anticipated that these morphogens also may stimulate the “redifferentiation” of committed cells.
Members of this morphogenic class of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also known as Vgr2), GDF-8, GDF-9, GDF-10, GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2 or BMP-13), GDF-7 (also known as CDMP-3 or BMP-12), the Xenopus homolog Vg1 and NODAL, UNIVIN, SCREW, ADMP, and NEURAL, to name but a few.
By way of illustration using exemplary family members, the tertiary and quaternary structure of both TGF-β2 and OP-1 have been determined. Although TGF-β2 and OP-1 exhibit only about 35% amino acid identity in their respective amino acid sequences the tertiary and quaternary structures of both molecules are strikingly similar. Both TGF-β2 and OP-1 are dimeric in nature and have a unique folding pattern involving six of the seven C-terminal cysteine residues. In each subunit four cysteines bond to generate an eight residue ring, and two additional cysteine residues form a disulfide bond that passes through the ring to form a knot-like structure. With a numbering scheme beginning with the most N-terminal cysteine of the 7 conserved cysteine residues assigned number 1, the 2nd and 6th cysteine residues bond to close one side of the eight residue ring while the 3rd and 7th cysteine residues close the other side. The 1 st and 5th conserved cysteine residues bond through the center of the ring to form the core of the knot. The 4th cysteine forms an interchain disulfide bond with the corresponding residue in the other subunit.
The TGF-β2 and OP-1 monomer subunits comprise three major structural elements and an N-terminal region. The structural elements are made up of regions of contiguous polypeptide chain that possess over 50% secondary structure of the following types: (1) loop, (2) α-helix and (3) β-sheet. Furthermore, in these regions the N-terminal and C-terminal strands are not more than 7 Å apart. The residues between the 1st and 2nd conserved cysteines form a structural region characterized by an anti-parallel β-sheet finger, referred to herein as the finger 1 region (F1). Similarly the residues between the 5th and 6th conserved cysteines also form an anti-parallel β-sheet finger, referred to herein as the finger 2 region (F2). A β-sheet finger is a single amino acid chain, comprising a β-strand that folds back on itself by means of a β-turn or some larger loop so that the entering and exiting strands form one or more anti-parallel β-sheet structures. The third major structural region, involving the residues between the 3rd and 4th conserved cysteines is characterized by a three turn α-helix referred to herein as the heel region (H). In the dimeric forms of both TGF-β2 and OP-1, the subunits are oriented such that the heel region of one subunit contacts the finger regions of the other subunit with the knot regions of the connected subunits forming the core of the molecule. The 4th cysteine forms a disulfide bridge with its counterpart on the second chain thereby equivalently linking the chains at the center of the palms, as further described herein below. The dimer thus formed is an ellipsoidal (cigar shaped) molecule when viewed from the top looking down the two-fold axis of symmetry between the subunits.
Whether naturally-occurring, or recombinantly or synthetically prepared, true morphogens within the TGF-β superfamily, such as osteogenic proteins, can induce recruitment and stimulation of progenitor cells, thereby inducing their differentiation, e.g., into chondrocytes and osteoblasts, and further inducing differentiation of intermediate cartilage, vascularization, bone formation, remodeling, and, finally, marrow differentiation. Numerous practitioners have demonstrated the ability of osteogenic proteins, when admixed with either naturally-sourced matrix materials such as collagen or synthetically-prepared polymeric matrix materials, to induce true bone formation, including endochondral bone formation, under conditions where true replacement bone would not otherwise occur. For example, when combined with a matrix material, these osteogenic proteins induce formation of new bone in large segmental bone defects, spinal fusions, and fractures, to name but a few.
Bacterial and other prokaryotic expression systems are relied on in the art as preferred means for generating both native and biosynthetic or recombinant proteins. Prokaryotic systems such as E. coli are useful for producing commercial quantities of proteins, as well as for evaluating biological and chemical properties of naturally occurring or synthetically prepared mutants and analogs. Typically, an over-expressed eukaryotic protein aggregates into an insoluble intracellular precipitate (“inclusion body”) in the prokaryote host cell. The aggregated protein is then collected from the inclusion bodies, solubilized using one or more standard denaturing agents, and then allowed, or induced, to refold into a functional state.
Optimal refolding requires proper formation of any disulfide bonds to form and stabilize a biologically active protein structure. However, not all naturally-occurring proteins when solubilized readily refold. Despite careful manipulation of the chemistries for refolding, the yields of optimally folded, stable and/or biologically active protein remain low. Many of the aforementioned proteins, including BMPs, fall into the category of poor refolder proteins. For example, while some members of the BMP protein family can be folded relatively efficiently in vitro as, for example, when produced in E. coli or other prokaryotic hosts, many others, including BMP-5, BMP-6, and BMP-7, are not. See, e.g., EP 0433225, U.S. Pat. Nos. 5,399,677, 5,756,308, and 5,804,416.
A need remains for improved means for designing and successfully producing recombinant, chemosynthetic and/or biosynthetic members of the TGF-β superfamily of proteins, including morphogenic proteins.